Neuronal apoptosis occurs during normal nervous system development and in a variety of neuropathological processes including hypoxic/ischemic injury and neurodegenerative diseases. In many cell types, including neurons, apoptotic death can be inhibited by the proto-oncogene bcl-2. Bcl-2 is expressed in subpopulations of cells in the embryonic nervous system but is virtually absent in the adult brain, suggesting that it plays a role in developmentally-regulated neuronal apoptosis. Surprisingly, targeted disruption of the bcl-2 gene in mice did not lead to an obvious nervous system abnormality. This finding may be attributed to functional redundancy, as bcl-2 is a member of a family of related genes which includes bcl-x. Unlike Bcl-2, Bcl-x is present at high levels in both embryonic and adult nervous systems. To determine the function of bcl-x in the nervous system, we have examined the effect of a targeted disruption of the bcl-x gene. Homozygous mutant mice died around embryonic day 13 (E13) from a failure to generate mature red blood cells. Mice analyzed prior to E13 exhibited extensive apoptosis of postmitotic immature neurons. The neuronal apoptosis was not secondary to hypoxic damage since it occurred prior to the defect in hematopoiesis and was seen in double knockout chimeric mice that suffered no hypoxic damage. Our preliminary studies of bcl-x- deficient primary cell cultures from the E12.5 telencephalon indicate increased apoptosis and decreased immature neuron survival compared to cultures from heterozygote and wild-type mice. Apoptosis of bcl-x- deficient immature neurons was inhibited by serum growth factors and insulin, but not by a protein synthesis inhibitor. Mature bcl-x-deficient neurons were generated in growth factor containing medium and showed an increased vulnerability to subsequent serum deprivation. The goals of this grant proposal are to define the roles of bcl-x in neuronal development and neuropathology using a multi-faceted analysis of the bcl-x deficient nervous system. First, in vivo studies will define the relationship of bcl-x-dependent neuronal apoptosis to the cell cycle, the complementary roles of bcl-x and bcl-2 in neuronal apoptosis, and the possible role of bcl-x in peripheral nervous system development. Second, in vitro studies of telencephalic neurons will determine the ability of neurotrophic and growth factors to affect neuron survival in bcl-x, bcl-2 and bcl-x/bcl-2-deficient cells. Finally, the cellular mediators of bcl-x action during nervous system development and the possible involvement of bcl-x in an in vitro model of neurotoxicity will be explored. These investigations should yield important data on bcl-x-dependent and independent anti-apoptotic pathways in neurons and provide insights into the regulation of neuron survival during normal development and in neuropathologic conditions.